Optical indicators for ions are important for qualitative and quantitative determination of ions, particularly in living cells. Fluorescent indicators for alkali metal ions, in particular Na.sup.+ and K.sup.+, permit the continuous or intermittent optical determination of these ions in living cells. Such indicators are also useful for measuring ions in extracellular spaces; in vesicles; in vascular tissue of plants and animals; biological fluids such as blood and urine; in fermentation media; in environmental samples such as water, soil, waste water and seawater; and in chemical reactors.
Fluorescent indicators are already known for a variety of ions, particularly for biologically important ions such as calcium, magnesium, sodium and potassium. See e.g. Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, Part IV, Set 22 (5th ed. 1992). For most biological applications, it is essential that the indicators be effective in aqueous solutions. It is also important that indicators for biological applications be insensitive to pH changes over the physiological range (pH 6-8) and sensitive to ion concentrations in the physiological range (for sodium, a K.sub.d of about 5 mM to about 20 mM). It is also beneficial if the indicator absorbs and emits light in the visible spectrum where biological materials have low intrinsic absorbance or fluorescence.
Benzofuranyl indicators for alkali metal ions such as Na.sup.+, K.sup.+, and Li.sup.+ are described by Tsien, et al. in U.S. Pat. No. 5,134,232 (1992) for intracellular use. In particular, Tsien, et al. describe fluorescent indicator compounds containing an aza crown ether attached to one or two fluorophores. The preferred compounds of Tsien, et al. (e.g. SBFI, SBFO, SBFP) contain two benzofuranyl fluorophores that contain an additional carboxy-substituted aromatic substituent on the fluorophore.
Although the benzofuranyl indicators described as preferred by Tsien, et al. have improved sensitivity to Na.sup.+ and K.sup.+ over previously known materials, including other materials claimed in the patent, the preferred indicators are not without disadvantages. The benzofuranyl indicators, particularly SBFO, tend to leak from cells, limiting their effectiveness in intracellular applications. The benzofuranyl indicators are also limited because they are only excitable in the UV (below about 380 nm) rather than in the visible range. Use of a UV excitation wavelength usually results in more background signal from contaminants in the sample and a greater likelihood of phototoxicity to living cells. In addition, when an indicator is incorporated in a fiber optic sensor, the transmittance of light in a fiber optic is significantly greater at visible wavelengths than in the ultraviolet. Furthermore, benzofuranyl indicators with shorter wavelength excitation cannot be used optimally with instruments such as flow cytometers and confocal microscopes that use the argon laser for excitation (at 488 nm).
A fluorescent N-phenyl-monoaza crown ether containing a benzoxazinone fluorophore (BOZ-crown) or a merocyanine laser dye (DCM-crown) is described by Fery-Forgues, et al., NEW J. CHEM 14, 617 (1990). The fluorescent monoaza crown ethers have absorption maxima in acetonitrile of 490 nm and 464 nm respectively. Like the materials of Tsien, et al., the fluorophore is linked to the crown phenyl substituent by a trans-ethylenic linkage, except that it is rotatable instead of fixed. Unlike the materials described by Tsien, et al., however, these compounds cannot be used in aqueous environments because even minute amounts of water appear to cause a shift to non-linearity in the fluorescence response to ion binding. Furthermore, this type of fluorophore typically has a low fluorescence yield in water and the compounds described have low water solubility.
None of the crown ether compounds described above are conjugated to other materials that assist in localizing or retaining the indicators inside the cell, or used for preparing conjugates with polymers. There is a need for fluorescent indicators for alkali-metal ions that can be attached to polymers for use in remote sensing of ions or enhancing the solubility or localization of the optical sensor. The advantage of conjugation of the indicator to water-soluble polymers to improve retention of other ion indicators in the cytosol has been described in Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, supra, but Haugland does not describe indicators for alkali-metal ions conjugated to polymers or other materials.
U.S. Pat. No. 5,133,934 describes a hydrophobic career incorporating a bis crown ether compound and a reporter substance. Although the patent mentions that fluorescent indicators can be used as reporter substances, the preferred reporter substance (the only one evaluated) is a nonfluorescent compound 7-decyl-MEDPIN. Other nonfluorescent crown ethers on a solid support have been described. The use of (allyloxy)methyl-substituted diaza crown ethers that have been covalently bonded to silica gel for separation of certain heavy metal ions, is described by Bradshaw, et al., J. ORG. CHEM. 53, 3190 (1988). Selective column concentration of alkali-metal ions using crown ethers is described by Hyashita, et al., ANAL. CHEM. 63, 1844 (1991).
There is a need for fluorescent ion indicators with desirable spectral properties, that also can be covalently coupled to polymers, lipids, or other solid phase materials or intracellular or extracellular components. There is need further for indicators that can function in aqueous solution and whose spectral properties facilitate detection in the visible range. The novel indicator compounds containing xanthylium fluorophores described herein differ from previously described materials in that they have improved spectral properties and sensitivity that enhance their use as indicators of trace amounts of biologically important ions of alkali-metals. The xanthylium indicators have a high absorbance in the visible spectrum (generally with an extinction coefficient of at least 60,000 cm.sup.-1 M.sup.-1 for indicators containing one xanthylium dye to greater than about 120,000 cm.sup.-1 M.sup.-1 for those containing two xanthylium dyes) and bright long wavelength fluorescence of the metal-indicator complex (generally a quantum yield of greater than about 0.2). Furthermore, they are useful in aqueous solutions and can be covalently reacted with a variety of materials. The covalent attachment as well as desirable spectral properties also make the materials useful in remote sensing devices, particularly where attachment to a solid phase is required.